The present invention relates to a method of reducing diketocarboxylic acids or hydroxyketo carboxylic acids or their esters to form hydroxyl compounds.
Optically active hydroxyl compounds are valuable chiral materials. Chiral diols are, for example, important raw materials for a multitude of active compounds in the pharmacy and in plant protection and also for catalysts.
For the manufacture of chiral compounds biotechnological processes can be used which utilize either whole microorganism cells or isolated enzymes.
Suitable enzymes for the synthesis of alcohols are, among others, oxidoreductases, which are described in EP 911 07 067.0 (produced from lactobacillus kefir), EP 0 796 691 A2 and PCT/DE99/00848 (from lactobacillus brevis). However, the syntheses require accessibility to the suitable enzymes, the addition of a soluble coenzyme (NADH, NADPH) and a coenzyme regeneration system. Likewise, in WO97/00968 the use of reductases for the reduction of keto groups is described.
In addition U.S. Pat. No 5,342,767 discloses a process, wherein a reduction is performed with the use of alcohol dehydrogenases from lactobacillus kefir.
DE 196 10 984.1 also discloses a method wherein an alcohol dehydrogenase is utilized. In addition to the purified enzymes also whole cells can also be utilized. The subject of this invention, however, does not relate to the reaction of compounds with two or more keto groups. The process is, furthermore, not concerned with the conversion of keto carboxylic acids and their esters.
Microorganisms present a cost-effective alternative to enzymes for reduction processes. However, these processes have often a low yield (F. Aragazzini et al., Appl. Microbiol. Biotechnol. (1986) 24, 175-177). Furthermore, yield-lowering side reactions often occur and the products do not always have a sufficient enantiomeric purity. The production yield and quality depend greatly on the strain used and on the growth conditions.
From the European patent application 0 569 998 A2 finally a method is known wherein various microorganisms are used for the reduction of diketoesters containing ether groups. The microorganisms usable herein include a large number of yeasts and bacteria, but no lactobacillus species.
It is accordingly the object of the present invention to provide a method for the reduction of diketo- or hydroxylketocarboxylic acids or the esters thereof, which does not have the disadvantages pointed out above. Under hydroxyl groups also hydroxyl groups are to be understood which are masked by protective groups.
In a method of reducing diketocarboxylic acids or hydroxyketocarboxylic acids or their esters, at least one keto group is converted to a hydroxyl group in the presence of lactobacillus species.
In a preferred embodiment, the reduction is performed so as to provide diols.
The method according to the invention includes in particular the catalytic reduction of the prochiral 3.5-dioxocarboxylic acid derivatives according to the formula 1: 
A, B=Cxe2x95x90O, CHOxcexa3, with xcexa3=H or a protective group for the hydroxyl function; wherein A and B may be identical or different.
R1, R2=H or a component of the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, aralkyl, cycloalkylalkyl, wherein the components may also be mono- or polysubstituted by heteroatoms, such as Si, N, P, O, S, F, Cl, Br, or I or they may be completely replaced by heteroatoms.
R3=H, metal cations or a component of the group alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, aralkyl, cycloalkylalkyl, wherein the components may be mono or polysubstituted by hetero atoms, such as, for example, Si, N, P, O, S, F, Cl, Br, or I.
Y=a component of the group comprising alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, aralkyl, cycloalkylalkyl, wherein the components may be mono- or polysubstituted by heteroatoms such as, for example, Si, N, P, O, S, F, Cl, Br, or I. Excluded is Xxe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94 wherein X=alkyl, cycloalkyl, aryl, aralkyl, cycloalkylalkyl. Among the halogens, fluorine and chlorine are particularly preferred. n=O-10.
Alkyls are considered to be straight-chain as well as branched saturated carbon chains. Methyl, ethyl, n-propyl, i-propyl, t-butyl, pentyl, i-pentyl, n-hexyl, i-hexyl, may be mentioned as examples.
Alkynyl comprises straight-chain and branched unsaturated hydrocarbons, which contain at least one xe2x80x94Cxe2x89xa1Cxe2x80x94 band such as for example ethynyl or propynyl.
Cycloalkyl comprises saturated annular hydrocarbon chains which consist of three, four, five, six or seven hydrocarbon atoms.
Cycloalkenyl designates annular hydrocarbons with five, six, seven or eight carbon atoms.
Aryl comprises aromatic systems including heteroaromats and subsituted aromatic systems such as for example phenyl, p-methylphenyl or furanyl.
Aralkyl are aryl rests which are bound by way of alkyl groups, for example benzyl rests.
Cycloalkylalkyl comprises cycloalkyl radicals which are bonded by way of alkyl groups.
Also, reactions of the compounds according to formulas 2, 3, and 4 are possible. 
Herein, R1, R2, R3 and Y have the same meaning as in formula 1.
xcexa3=H or protective groups for the hydroxyl functions.
With the method according to the invention, it is therefore possible to produce 3,5 dihydrocarboxylic acid derivatives as represented by the following formula 5. 
Herein R1, R2, R3 and Y have the same meaning as in formula 1.
xcexa3=H or protective groups for the hydroxyl function.
In contrast to the state of the art, with the method according to the invention compounds according to the formulas 3 and 4 can be converted as substrates.
Depending on the configurations at the stereocenters C-3 and C-5, the 3,5-dihydroxycarboxylic acid derivatives of the formula 5 prepared in accordance with the invention can be utilized in a controlled manner during the synthesis of chiral natural substances, pharmaceutical and agricultural active compounds, catalysts and inhibitors. Examples, herefor are HMG-CoA reductase inhibitors of the mevinic acid type and the lipase-inhibitors of the lipstatin-type.
Other natural substances or effective compounds require other configurations of the stereogenic centers in position C-3 and C-5. This is also possible with the present invention.
The method according to the invention facilitates, during the reduction of 3,5-dioxocarboxylic acid derivatives, the area selective introduction of a hydroxyl group in position C-3 or C-5 or C-3 and C-5, wherein a product with a r-configuration is obtained.
Concerning the designation r-configuration, the following is pointed out as an example with respect to formula 3:
In the formula 3, the Oxcexa3-group in the 5-position projects from the paper plane whereas Y and the carbon atoms of the basic carbon structure are disposed in the plane of the paper: 
This configuration will be designated below as the r-configuration independently of Y.
Furthermore, the method according to the invention facilitates during the reduction of 3,5 dioxycarboxylic acid derivatives of the formula 2, a specific fixing of the stereo centers in the positions C-2 and C-4. This is particularly true for a case in which R1 and R2 differ from H, and R1 represents for example a methyl group. The enantiomeric purity as well as the diastereomeric purity are very high in that case ( greater than 95%).
In accordance with the invention also mixtures can be produced with the reduction method, which include compounds with different hydroxyl group contents. Examples herefor are mixtures, which consist of compounds of the formulae 3, 4, and 5, wherein R1, R2=H; Y=xe2x80x94CH3 or xe2x80x94CH2Cl and R3=C(CH3)3.
The method according to the invention is performed with Lactobacillus types. Basically, any Lactobacillus type may be used. Particular preference however is given to the use of Lactobacillus kefir and Lactobacillus brevis. 
The method according to the invention can be performed under customary fermentation conditions and in the customary reactors.
As co-substrate, a carbon-containing substrate, which can easily be metabolized, such as glucose, may be used. In this way, the reduction equivalents, which are consumed during the conversion, can be replaced.
The reactions are performed in accordance with the invention at temperatures of 10 to 50xc2x0 C., preferably at 15-40xc2x0 C.
The pH value is between 2 and 10, preferably between 4 and 8. To ensure a suitable pH value, any buffer substance common in fermentation tchnology may be used. These are for example triethanolamine, phosphate buffers, phosphate-citrate-buffers, 2-amino-2-(hydroxymethyl)-1,3-propandiol-buffers, 2[N-morpholino] ethanesulfonic acid buffers (MES) or tris buffers. The concentration ranges for the buffers are preferably between 50 and 500 mmol/l.
As reactors any known reactor type may be used. For example, conventional stirred reactors as well as fixed bed reactors can be employed.
With the method according to the invention, racemate separations or diastereomer separations, which are costly and may detrimentally affect the environment""can be omitted. The binding and cleavage of a stoichiometric amount of a homochiral auxiliary group which is required in a diastereoselective synthesis, is avoided. Furthermore, the carbon structure of the dihydroxycarboxylic acid esters is complete already in the initial compounds that is, the stereocenters are introduced into the overall synthesis sequence only at a later point in time. In this way, the loss of homochiral material is kept low. At the same time, very high stereo selectivities are achieved with the method according to the invention. With the use of whole cells, there is also no need to introduce expensive coenzyme and coenzyme regeneration systems into the reaction.
The compounds according to formulas 1 to 5 made in accordance with the invention can be used particularly for the production of chiral natural substances, pharmaceutical and agricultural active compounds, catalysts and inhibitors. Examples herefor are HMG-CoA reductase inhibitors of the mervinic acid type and lipase-inhibitors of the lipstatin type.
Below, the invention will be described in greater detail on the basis of examples.